Electrochemical sensors membrane

ABSTRACT

A solid state, multi-use electrochemical sensor having an electrically nonconductive substrate, a working electrode, and a semi-permeable membrane covering the working electrode. The working electrode includes an electrically conductive material adhered to a portion of the substrate. A first portion of the conductive material is covered with an electrically insulating dielectric coating, and a second portion of the conductive material is covered with an active layer. The active layer includes a catalytically active quantity of an enzyme carried by platinized carbon powder particles, which are distributed throughout the active layer. A sensor package for incorporating a sensor is provided.

This is a divisional of application Ser. No. 08/266,824 filed on Jun.27, 1994, now U.S. Pat. No. 5,494,562.

The present invention relates generally to electrochemical sensors and,more particularly, to enzyme catalyzed electrochemical sensors includingglucose and lactate sensors. Novel packages incorporating enzymeelectrodes having an enzyme contained in an electrically conductivesubstrate, responding to the catalytic activity of the enzyme in thepresence of the substrate are described.

TECHNICAL REVIEW

The concentration of glucose and lactate in the blood is extremelyimportant for maintaining homeostasis. For example, a concentration ofglucose below the normal range, or hypoglycemia, can causeunconsciousness and lowered blood pressure, and may even result indeath. A concentration of glucose at levels higher than normal, orhyperglycemia, can result in synthesis of fatty acids and cholesterol,and in diabetics, coma. The measurement of the concentration of glucosein a person's blood, therefore, has become a necessity for diabetics whocontrol the level of blood glucose by insulin therapy.

In a clinical setting, accurate and relatively fast determinations ofglucose and/or lactate levels can be determined from blood samplesutilizing electrochemical sensors. Conventional sensors are fabricatedto be large, comprising many serviceable parts, or small, planar-typesensors which may be more convenient in many circumstances. The term"planar" as used herein refers to the well-known procedure offabricating a substantially planar structure comprising layers ofrelatively thin materials, for example, using the well-known thick orthin-film techniques. See, for example, Liu et al., U.S. Pat. No.4,571,292, and Papadakis et al., U.S. Pat. No. 4,536,274, both of whichare incorporated herein by reference.

In the clinical setting, it is a goal to maximize the data obtainablefrom relatively small test sample volumes (microliters) during chemicalblood analysis. Fabrication of a sensor sample chamber for holding ablood sample in contact with a sensor is desirable in this regard sothat many determinations may be simultaneously performed on a testsample, for example, using a series of interconnected sensors, eachconstructed to detect a different analyte, from a small test samplevolume. However, as a sample chamber is made smaller, the concentrationof contaminants in a sample, as those released from sensor componentsthemselves, especially components defining the sample chamber, and/orcertain reaction products of the sensor itself is increased. Suchcontamination may result in premature sensor failure.

There are two major types of glucose or lactate electrode sensors. Thefirst is an electrocatalytic device which utilizes direct oxidation ofglucose or lactate for obtaining a measurable response. The second is anenzyme electrode which utilizes an enzyme to convert glucose or lactateto an electroactive product which is then analyzed electrochemically.

With respect to glucose sensors, the latter type of electrode sensors,including an enzyme electrode, converts glucose in the presence ofenzymes, such as glucose oxidase, and results in the formation ofreaction products including hydrogen peroxide according to the followingreactions: ##STR1##

In these reactions, glucose reacts with oxygen to form gluconolactoneand hydrogen peroxide. A suitable electrode can then measure theformation of hydrogen peroxide, as an electrical signal. The signal isproduced following the transfer of electrons from the peroxide to theelectrode, and under suitable conditions the enzyme catalyzed flow ofcurrent is proportional to the glucose concentration. Lactate electrodesensors including an enzyme electrode, similarly convert lactate in thepresence of enzymes, such as lactate oxidase.

Numerous devices for determination of glucose and lactate have beendescribed, however most of them have some limitation with respect toreproducibility, speed of response, test same volume, number ofeffective uses, and the range of detection. Some existing commercialmethods rely on utilization of hydrogen peroxide measurement as outlinedabove.

With respect to glucose sensors, in known enzyme electrodes, glucose andoxygen from blood, as well as some interferants, such as ascorbic acidand uric acid diffuse through a primary membrane of the sensor. As theglucose, oxygen and interferants reach a second membrane, an enzyme,such as glucose oxidase, catalyzes the conversion of glucose to hydrogenperoxide and gluconolactone. The hydrogen peroxide may diffuse backthrough the primary membrane, or it may further diffuse through thesecondary membrane to an electrode where it can be reacted to formoxygen and a proton to produce a current proportional to the glucoseconcentration. The electrode's membrane assembly serves severalfunctions, including selectively allowing the passage of glucosetherethrough, providing a location between the primary and secondarymembranes for an enzyme to catalyze the reaction between the glucose andoxygen passing through the primary membrane, and allowing only hydrogenperoxide through the secondary membrane to the electrode.

A single-layered electrode membrane was described by Jones in EP PatentNo. 207 370 B1. This reference is directed to an electrochemical sensorincluding three primary components: a metal electrode, a reactive layerof immobilized enzyme directly on an anode, and a single-layeredmembrane. The membrane disclosed in EP 207,370 B1, is glucose permeableand whole blood compatible, thereby eliminating the need for thesecondary membrane typical in prior art sensors. The membrane is formedfrom a dispersion of a polymerizable silicon-containing compound appliedin an incompletely cured form, having a liquid carrier which isessentially insoluble in the dispersed phase and removable from thedispersion during curing. The membrane cures as a continuous layer, filmor membrane, having high glucose permeability.

It has been found, however, that the single membrane layer disclosed inEP 207,370 B1 prevents only anionic interfering substances, such asascorbic acid and uric acid, from passing therethrough. Neutral species,such as acetaminophen, can diffuse through the membrane and influencethe sensor's sensitivity and accuracy.

As noted above, enzyme electrodes convert glucose into hydrogenperoxide, which can be reacted to produce a current proportional to theglucose concentration. Enzyme electrodes adapted to measure otheranalytes have also been described in the art. An enzyme electrode havingan electrically conductive support member which consists of, orcomprises, a porous layer of resin-bonded carbon or graphite particlesis disclosed by Bennetto et al., in U.S. Pat. No. 4,970,145. The carbonor graphite particles have a finely divided platinum group metalintimately mixed therewith, to form a porous, substantially homogeneous,substrate layer into which the enzyme is adsorbed or immobilized. Thepreferred substrate materials are resin bonded, platinized carbon paperelectrodes, comprising platinized carbon powder particles bonded onto acarbon paper substrate using a synthetic resin, preferablypolytetrafluoroethylene, as the binder. These electrode materials aremanufactured by depositing colloidal size particles of platinum,palladium, or other platinum group metal, onto finely divided particlesof carbon or graphite, blending the platinized or palladized carbon orgraphite particles with a fluorocarbon resin, preferablypolytetrafluoroethylene, and applying the mixture onto an electricallyconductive support, such as carbon paper, or a filamentous carbon fiberweb.

The above-referenced enzyme electrodes require premolding of thegraphite or carbon base often under conditions requiting sintering ofthe molded compact to fuse the binder, which, as noted, is a highmelting point hydrophobic synthetic resin. These high temperatures woulddestroy enzymes, such as glucose oxidase or lactate oxidase.

Enzyme electrodes comprising an enzyme or mixture of enzymes immobilizedor adsorbed onto a porous layer of resin bonded platinized or palladizedcarbon or graphite particles without a high temperature binder have beendisclosed by Mullen, in U.S. Pat. No. 5,160,418. Mullen disclosed thatthe high temperature binders can either be dispensed with entirely orreplaced by a low temperature, preferably water soluble or waterdispersible binder, such as gelatin (a binder which can be activated atroom temperature, which does not require high temperature sintering).

Despite the above improvements in the art, however, a need remains foraccurate, multi-use glucose and lactate sensors, incorporating a glucoseor lactate and oxygen-permeable membrane and an enzyme electrode. Inaddition, there is a need for an electrochemical sensor package whichcan be used with a small blood sample and for extended sampling or usesin a clinical setting. An electrochemical sensor of this type that doesnot require maintenance, i.e. remembraning, electrode cleaning, etc.would also be desired.

It is therefore an object of the present invention to provide animproved electrochemical sensor, and method of making the same,generally incorporating enzyme electrodes having a metallized carbonbase and an overlying silicon-containing protective glucose and/orlactate permeable membrane.

It is a further object of the invention to provide an electrochemicalsensor incorporating an interference correcting electrode onto thesensor to provide efficiency over extended sampling periods.

It is a further object of the present invention to provide an improvedsensor package, which can be used with a series of interconnectedsensors, including a small sample chamber.

It is a still further object of this invention to provide an improvementin a planar electrode comprising using a metallized carbon active layerover a metal contact, and having an outer membrane which enables rapidtesting of samples, including blood, to determine glucose and/or lactateconcentrations.

It is a still further object of this invention to provide a planarsensor having a small sample chamber which incorporates a velocitycompensator to allow fluid flow without incurring problems ofinsufficient wash-out, i.e. sample carryover, or velocity modificationin the chamber, thus enabling fast filling and emptying of the chamberand increasing sample throughput.

It is a still further object of this invention to provide means andmethod for attaching small resilient electrical leads to a plurality ofcontacts in an electrical sensor with positive predetermined positioningrapidly and efficiently and with high precision and accuracy.

It is still a further object of the invention to provide a method forpost-treating sensors to prolong the storage life or wet-up of thesensor.

It is still a further object of the invention to provide multi-useglucose and lactate sensors having a long life.

It is still a further object of the invention to provide a method offormulating an enzyme into a paste for use in an electrode.

It is still a further object of the invention to provide a method offormulating cellulose acetate into a paste for use in an electrode.

Accordingly, the present invention provides a solid state, planarelectrochemical sensor including an electrically nonconductivesubstrate, a working electrode, and a semi-permeable membrane coveringthe working electrode, which permits glucose and oxygen or lactate andoxygen to pass through to the electrode. The working electrode includesan electrically conductive material adhered to a portion of thesubstrate. A first portion of the conductive material is covered with anelectrically insulating dielectric coating, and a second portion of theconductive material is covered with an active layer. The active layerincludes a catalytically active quantity of an enzyme, such as glucoseoxidase or lactate oxidase, carried by platinized carbon powderparticles, which are distributed throughout the active layer.

The sensor may further include a counter electrode having a secondelectrically conductive material adhered to a second portion of thenonconductive substrate. A portion of the second conductive material iscovered with the electrically insulating dielectric coating, and atleast one portion of the second conductive material remains uncovered.

In one embodiment of the present invention, the nonconductive substrateis made from alumina admixed with a glass binder, and the conductivematerials are thick-film pastes of either silver, gold, or platinum. Thedielectric coating is made from either ceramics, glasses, polymers, orcombinations thereof. The semi-permeable membrane can be formed fromcellulose acetate, polyurethane, silicone compounds, and other materialsknown in the art such as Nafion® material available from E. I. DuPont deNemours & Co., Wilmington, Del. The preferred membrane is a dispersionof a polymerizable silicon-containing compound applied in anincompletely cured form of a silicone compound dispersed phase in aliquid carrier. The semi-permeable membrane includes a silicone compoundhaving at least about 10.0 percent colloidal silica, by weight. Thepreferred membrane includes at least about 14.0 percent colloidalsilica, by weight.

In another embodiment of the present invention, the electrochemicalsensor may further include a reference electrode including a thirdelectrically conductive silver material adhered to a third portion ofthe substrate. A first portion of the third conductive material iscovered with the electrically insulating dielectric coating, and asecond portion of the third conductive material remains uncovered by theelectrically insulating dielectric coating. The third electricallyconductive material typically includes a silver/silver chloridethick-film paste. The second portion of the conductive material iscovered by cellulose acetate.

In still another embodiment of the present invention, theelectrochemical sensor may further include an interference correctingelectrode including a fourth electrically conductive material adhered toa fourth portion of the substrate. A first portion of the conductivematerial is covered with the electrically insulating dielectric coating,and a second portion of the conductive material is covered with aninactive layer. The inactive layer includes an inactive proteinimmobilized onto platinized carbon powder particles, which aredistributed substantially uniformly throughout the inactive layer.

In another embodiment of the present invention, the semi-permeablemembrane is post-treated with a high boiling point, water soluable,hydrophilic liquid anti-drying agent.

In a further aspect of the present invention, an electrochemical sensorpackage is provided. The package includes a housing having a recess witha perimeter and at least one passageway connected to the recess. Agasket contacts the recess perimeter and a solid state, planarelectrochemical sensor, as described above, and forms a sealtherebetween. The housing and electrochemical sensor define a samplechamber.

In yet another embodiment of the sensor package of the presentinvention, the package further includes a contact lead frame. The leadframe includes leads secured to the frame at a first end, and a recessfor the sensor at the opposite end. The contact lead frame may furtherinclude a stabilizer bar for aligning the leads with contact pads on thesurface of the sensor. A groove may also be provided in the package forreceipt of the stabilizer bar as the leads are wrapped over the topportion of the lead frame to be aligned with the sensor contact pads. Apad, or the like, may also be provided in the package of the presentinvention for supporting the sensor in the recess of the contact leadframe.

In another embodiment of the present invention, the sensor packageincludes a velocity compensator or bump within the sample chamber. Thevelocity compensator can be a molded part of the housing and preferablyfaces the sensor.

The method of forming a solid state, planar electrochemical sensorincludes selecting a suitable substrate material made from electricallynonconductive material, and forming it into a desired shape and size. Anelectrically conductive material is then deposited onto a portion of thesubstrate. Next, a portion of the conductive material is covered with anelectrically insulating dielectric coating, and a portion of theconductive material is uncovered so as to define an electrode area. Aworking electrode is then formed on the electrode area which includes anactive layer comprising a catalytically active quantity of an enzyme,such as glucose oxidase or lactate oxidase, immobilized onto platinizedcarbon powder particles, which are distributed substantially uniformlythroughout the active layer. Lastly, a semi-permeable membrane coversthe working electrode, which permits glucose and oxygen or lactate andoxygen to pass through to the electrode.

A preferred solid state, planar electrochemical sensor of the presentinvention is formed by selecting a suitably sized and shaped substratemade of an electrically nonconductive material, such as a ceramicmaterial comprising alumina and a glass binder. Four conductive stripsare deposited on top of the substrate so as to extend from a first endto a second end thereof. At the first end, the conductive strips definecontact pads for electrical connection, and at the opposite end of thesubstrate the strips define an electrode area for test sample exposure.The conductive strips may be deposited using thin or thick-filmsilk-screening techniques using conductive metal pastes of eithersilver, gold, and/or platinum. An electrically insulating dielectriccoating is similarly deposited on top of portions of the conductivestrips, while leaving portions of the strips uncovered to define thereference electrode, counter electrode, working electrode, interferencecorrecting electrode, and contact pads. The reference electrode isformed by depositing a layer of silver/silver chloride onto the exposedelectrode region. A cellulose acetate layer is then applied over thesilver/silver chloride reference electrode to protect the silverchloride from contaminants that would shift the reference potential.

A working electrode is formed by depositing an active layer, comprisinga catalytically active quantity of an enzyme immobilized onto toplatinized carbon powder particles, upon a conductive strip usingsimilar screen priming techniques. An interference correcting electrodeis formed in a manner similar to the working electrode. The interferencecorrecting electrode, however, includes an inactive layer, comprising aninactive protein in place of the catalytically active quantity of anenzyme immobilized onto platinized carbon particles. The interferencecorrecting electrode serves to adjust for electrochemically activeneutral species which may diffuse through a semi-permeable covermembrane, which is preferably spun-cast over the electrodes.

In an improvement provided by the present invention, a planar electrodefor use in glucose and/or lactate determinations, in vitro, has aninsulating base layer, a conductive layer, an overlying active layer andan outer protective membrane permeable to glucose and/or lactate. Theimprovement of the invention includes the active layer having an enzymereactive with one of glucose or lactate, and a platinized carbon powderparticle portion, so that the active layer is capable of causingformation of hydrogen peroxide in amounts proportional to the amount ofthe glucose or lactate being tested when they are exposed to the activelayer, and the outer protective membrane which is a silicone compoundhaving an additive incorporated therein for enabling transport ofglucose or lactate therethrough to enable rapid and accuratedeterminations of glucose or lactate.

In another improvement of this invention, a multi-use electrochemicalsensor is provided having a long life of effective use withoutmaintenance.

In another improvement of this invention, in a planar sensor having aplurality of electrodes positioned in a sample chamber with the samplechamber having a flow-through path, an inlet and an outlet, each havinga cross-sectional area less than the cross-sectional area of a portionof the chamber, a velocity compensator is provided. The velocitycompensator is a structural barrier mounted in the flow path between theinlet and outlet to reduce the cross-sectional area of the chamber inthe flow path so as to prevent contaminants from collecting, and tosubstantially maintain stability in fluid velocity when flowing throughthe chamber. The velocity compensator is preferably integral with thesample chamber and extends towards the electrodes without obstructingfluid flow over the electrodes.

In still another improvement in an electrochemical sensor mounted in ahousing and having a plurality of electrical contacts spaced close toeach other and a plurality of elongated axially extending electricalleads connected to the contacts, the leads are spaced apart by astabilizer bar. The stabilizer bar is attached to the leads andpositively positions the leads to establish electrical contact. Theleads are resilient and urged into contact by the stabilizer bar whichis preferably mounted on a lead frame base.

Other features of the present invention will become apparent from thefollowed detailed description when taken in connection with theaccompanying drawings. It is to be understood that the drawings aredesigned for the purposes of illustration only and are not intended as adefinition of the limits of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other features, objects and advantages of the presentinvention will be better understood from the following specificationwhen read in conjunction with the accompanying drawings, in which:

FIG. 1 is a perspective view of an electrochemical sensor package of thepresent invention;

FIG. 2 is an exploded view of the components of the sensor package shownin FIG. 1;

FIG. 3 is a cross-sectional side view of a contact lead frame shown inFIG. 2, with its leads partially open, taken along section line 3--3;

FIG. 4 is a cross-sectional side view of the contact lead frame shown inFIG. 3, with its leads wide open;

FIG. 5 is a magnified partial view of a sample chamber of the sensorpackage shown in FIG. 2;

FIG. 5A is a graphical illustration of the cross-sectional area of thesample chamber shown in FIG. 5 versus the position along the chamberflow path, with and without a velocity compensator (bump);

FIG. 5B is a graphical illustration of the ratio of sensing area to flowpath cross-sectional area of the sample chamber shown in FIG. 5 versusthe position along the chamber flow path, with and without the velocitycompensator;

FIG. 6 is a cross-sectional side view of the sample chamber shown inFIG. 5, taken along section line 6--6;

FIG. 6A is a cross-sectional side view of the sample chamber shown inFIG. 5 taken along section line 6--6, showing the velocity of fluid flowwith the velocity compensator (velocity: white>black);

FIG. 6B is a cross-sectional side view of the sample chamber shown inFIG. 5 taken along section line 6--6, showing the velocity of fluid flowwithout the velocity compensator (velocity: white>black).

FIG. 7 is a cross-sectional side view of the sample chamber shown inFIG. 6, taken along section line 7--7;

FIG. 8 is a cross-sectional side view of the sample chamber shown inFIG. 5, taken along section line 8--8;

FIG. 9A is a magnified top plan view of a sensor used in the sensorpackage shown in FIG. 1;

FIG. 9B is a magnified top plan view of another embodiment of a sensorused in the sensor package shown in FIG. 1;

FIG. 10 is a cross-sectional side view of a working electrode used inthe sensor shown in FIG. 9A, taken along section line 10--10;

FIG. 11 is a cross-sectional side view of a reference electrode used inthe sensor shown in FIG. 9A, taken along section line 11--11;

FIG. 12 is a graphical illustration of a glucose sensor response toglucose concentration in whole blood samples according to one embodimentof the present invention;

FIG. 13 is a graphical illustration of a lactate sensor response tolactate concentration in whole blood samples according to one embodimentof the present invention;

FIG. 14 is a graphical illustration of the effect of an interferencecorrecting electrode according to one embodiment of the presentinvention, as glucose concentrations are measured with and without thecorrecting electrode applied;

FIG. 15 is a graphical illustration of a glucose sensor output over anextended period of time and sample use;

FIG. 16 is a graphical illustration of glucose sensors response toglucose concentration, with and without a surfactant post-treatment;

FIG. 17 is a graphical illustration of glucose sensors response toglucose concentration, with a variety of surfactant post-treatments;

FIGS. 18A and 18B are graphical illustrations of glucose sensorsresponse to glucose concentration, after one week in storage at roomtemperature, with and without a surfactant post-treatment;

FIG. 19 is a graphical illustration of glucose sensors response toglucose concentration, and the effect of membrane thickness on thelinearity of the response;

FIG. 20 is a graphical illustration of glucose sensors response toglucose concentration, and a comparison between 2-layer and 4-layerspin-cast membranes on the linearity of the response;

FIGS. 21A and 21B are graphical illustrations of glucose sensorsresponse to glucose concentration, and a comparison between 2-layerspin-cast and stenciled membranes on the linearity of the response;

FIG. 22 is a graphical illustration of glucose sensors response toglucose concentration, and the effect of storage over an extended periodof time on the response when no surfactant is added to the platinizedactivated carbon of the active and inactive layers of the sensorelectrodes;

FIG. 23 is a graphical illustration of glucose sensors response toglucose concentration, and the effect of storage over an extended periodof time on the response when surfactant is added to the platinizedactivated carbon of the active and inactive layers of the sensorelectrodes;

FIG. 24 is a graphical illustration of glucose sensors response toglucose concentration, and the effect of adding a surfactant material tothe membrane material covering the sensor electrodes; and

FIG. 25 is a view of the steps of formation of a glucose sensor.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to the drawings, in which like reference numeralsdesignate like or corresponding parts throughout the several views, anassembled electrochemical sensor package 10 in accordance with apreferred embodiment of the present invention is illustrated in FIG. 1.Package 10 has a generally J-shaped body, including a handle portion 12,a main body 14, contact portion 16, and fluid or liquid passageway 18.

The internal components of sensor package 10 are shown in an explodedview in FIG. 2. Package 10 includes a J-shaped housing 20 having arecess 22 formed therein. The recess 22 which forms a part of a samplechamber 54 (FIG. 5) includes an outer perimeter 24 and at least onepassageway 18. Housing 20 has a substantially flat upper portion 120,sidewalls 122, 124, a frontal opening 126, and a rear wall 128 which iscontiguous with sensor package handle portion 12. Housing 20 furtherincludes a depressed inner rim 130 and projections 132 which contact thelead frame 32 when package 10 is assembled. A gasket 26 is provided tocontact, and form a seal between, the housing perimeter 24 and a sensor28. Gasket 26 is substantially rectangular-shaped and includes asubstantially oval-shaped opening 134, and two raised surfaces 136, 138which run along the length of gasket opening 134. Gasket raised portions136, 138 allow gasket 26 to fit around the housing recess perimeter 24,while also allowing recess 22 to be exposed to sensor 28. A sensor pad30 is provided to support sensor 28. Sensor pad 30 includes a series oftransverse protrusions 140 on rear side 142 which provide sensor 28 withadded support when package 10 is assembled. Lastly, a contact lead frame32 is provided to electrically connect sensor 28 to an instrument (notshown) which can measure and convert a current to determine analyteconcentration, i.e. glucose or lactate. Contact lead frame 32 includesfour leads 34 secured to a base 36 at a first end portion 38, and asensor recess 40 at a second end portion 42. The lead frame 32 can alsoinclude a stabilizer bar 44 for holding the leads in a predeterminedposition with respect to each other and aligning the leads 34 with thesensor 28. An additional recess 46 can be included for receipt ofstabilizer bar 44. It is also noted that an electrode O-ring 48,commercially available from Ciba Corning Diagnostics Corp. or the like,can be provided to maintain a seal between adjacent sensor packages, ora fluid conduit (not shown), each different sensor used tosimultaneously detect different analytes from the same fluid sample.

Referring now to FIGS. 3 and 4, a cross-sectional side view of contactlead frame 32 taken along section line 3--3, is shown with its leadspartially and wide open. As noted above, to provide an interface systembetween an instrument and contacts on the sensor 28 (described below),contact lead frame 32, including plural leads 34 secured to base 36 atend portion 38, is provided. Leads 34 are typically made from a highlyconductive, malleable metal, such as copper. Preferably, leads 34 aremade of a highly conductive, malleable material, which is beryllium,silver, gold, or platinum plated, due to the lower cost of platedmaterial. Most preferably, photo-etched, gold plated leads 34 are useddue to their high conductivity. The leads 34 are molded into one end 38of the contact lead frame 32. Lead frame 32 can be formed of anymaterial that is compatible with, and can be secured to, the sensorpackage housing 20. Typically, lead frame 32 is made from a rigid,durable material such as glass, ceramic, stainless steel, or a plasticmaterial such as acrylic, polyester, polycarbonate, polyvinyl chloride,and the like. Preferably, an acrylic plastic material, such as V825acrylic, available from Rohm & Haas Corp., Philadelphia, Pa., is used tomold lead frame 32 due to its strength, durability, relatively low costand ease of processing.

During assembly of the sensor package 10, sensor 28 is placed intorecess 40 and leads 34 are bent around the lead frame 32 until they makecontact with the sensor. Leads 34 contact the sensor with rounded springtips 50 which apply constant pressure on the sensor contacts. Stabilizerbar 44, which aligns the leads 34 with the sensor contacts, is securedin recess 46 after the leads are bent around the frame.

Preferably, the stabilizer bar, if present, is solvent cemented in placein recess 46. Instrument contact surfaces 52, which are exposed afterthe sensor package 10 is assembled, are formed as the leads are bentover the frame (as shown in FIG. 3). Once the leads are in contact withthe sensor, housing 20 is placed over the lead frame 32. The housing andlead frame are then secured together by being snap-fit, ultrasonicallywelded, adhesive bonded, or by other methods known to those skilled inthe art.

Referring now to FIGS. 5-8, a magnified view of a sample chamber 54 ofthe sensor package 10 is shown. As noted above, sample chamber 54 isdefined by the housing 20, the outer perimeter 24 around recess 22,gasket 26, and the sensor 28. At least one passageway 18, having aninlet 56 and an outlet 58, is provided to allow passage of a fluidsample, such as blood, into and out of sample chamber 54. Although, inthe embodiment illustrated, inlet 56 and outlet 58 pass through housing20, these openings can be formed in any manner to provide a passagewaythrough which a fluid sample could reach sample chamber 54. For example,openings, or channels, could be formed in the gasket 26, or otherpart(s) of the sensor package 10.

The sample chamber 54 of the present sensor package 10 also includes avelocity compensator 60 (Bump), which reduces the internal volume of thechamber and creates a cross-sectional area close to that of the inlet 56and outlet 58. FIG. 5A graphically illustrates the sample chamber 54cross-sectional area along the chamber flow path, with and withoutvelocity compensator 60. As shown in the graph, the cross-sectional areaof the sample chamber at the velocity compensator approaches that of theinlet and outlet. The velocity compensator or bump acts as a structuraldirector of fluid flow. Conventional sample delivery systems experienceproblems such as carryover of previous sample materials, and trapped airbubbles which are present or within the leading edge of the sample fluidto address a problem common to conventional sample delivery system.Typically, as a sample enters the chamber 54 its flow velocity abruptlyslows until the chamber is full. The sample velocity then increases toits initial level, leaving the solution at the chamber walls stagnant.Although sample chambers are washed between measurements, air bubblesand fluid can become trapped in the chamber in stagnant areas. These airbubbles and residual fluid effect the accuracy of the samplemeasurement. The velocity compensator 60 of the present invention,therefore, keeps the flow velocity stable within the chamber, andreduces or eliminates the stagnant areas where bubbles and fluid cancollect. Referring to FIGS. 6A and 6B, the velocity of fluid flowthrough the sample chamber is substantially uniform in the presence ofthe velocity compensator. In addition, velocity compensator 60 allowsthe use of a large sensing area with relatively small inlet 56 andoutlet 58 cross-sections. FIG. 5B graphically illustrates the ratio ofsensing area to flow path cross-sectional area along the chamber flowpath, with and without velocity compensator 60. Because thecross-sectional area of the chamber 54 is reduced (as shown in FIG. 5A)with the velocity compensator 60 in place, the ratio of sensing area toflow path is increased. This aspect of the present invention allowsfluid samples to more efficiently contact sensor 28 as they are passedthrough package 10. Moreover, by positioning the velocity compensator 60facing the sensor 28, samples are directed toward the sensor 28 whilebubbles are substantially eliminated.

FIG. 6 illustrates a cross-sectional side view of the sample chamber 54,taken along section line 6--6. The velocity compensator 60 is shown as amolded part of housing 20. Although shown having a rounded shape, avariety of smooth, sloped shapes, without stagnant areas, can be used.Furthermore, although shown as a molded part of the housing, velocitycompensator 60 can be a separate component, added to sample chamber 54.

Inlet 56 and outlet 58 portions are shown leading in and out of chamber54. These sample paths typically have diameters between about 0.02 inchand about 0.04 inch; preferably, the diameters are about 0.03 inch. Thesample chamber 54 has a sample diameter, with the velocity compensator60, of at least the size of the sample paths to about 0.06 inch. FIG. 8shows a side view of sensor package 10, taken along section line 8--8,through passageway 18. This view illustrates the relative sizes of thevelocity compensator 60, sample chamber 54, and passageway 18.

Housing 20, as well as inlet 56 and outlet 58, and velocity compensator60, can be fabricated from any material that is unreactive with a samplewhich passes into sample chamber 54 during analysis. For example,materials such as glass, ceramics, stainless steel, or plastic materialssuch as acrylic, polyester, polycarbonate, polyvinyl chloride, and thelike.

Preferably, a clear, transparent acrylic plastic material, such as V825acrylic from Rohm & Haas, is used to mold these parts due to itsstrength, durability, relatively low cost and ease of processing.

Gasket 26, shown in FIGS. 6 and 7, is typically formed from a materialwhich, when held firmly between recess perimeter 24 and sensor 28, formsa seal around sample chamber 54 through which the passage of fluids issubstantially prevented.

Typically, gasket 26 is formulated from a durable organic polymer whichdoes not creep or flow when stressed, has a low durometer rating, andcan be slightly hygroscopic. Preferably, a material used in thefabrication of gasket 26 has a hardness of between 10 and 100 on theShore A scale; more preferably, a hardness of from about 40 to about 70on the Shore A scale; and most preferably, a hardness of from about 45to about 55 on the Shore A scale.

Because gasket 26 is typically an organic polymer, it is fabricated soas not to contain a substantial amount of any mobile extractablematerials, such as plasticizers, which may leach into sensor 28.Additionally, as is the case for other sensor components as describedabove, it is important the material selected for formation of gasket 26be free of any species which could migrate into a sample in chamber 54,affecting electrochemical measurements, and/or destroying sensorcomponents. Material used in the formation of gasket 26 is preferablyselected to be essentially free of mobile transition and main groupmetals, especially battery metals such as iron, cobalt, nickel, lead,copper, extractables, and species such as sulfides which are deleteriousto preferred electrode materials.

Gasket 26 is typically formed form a highly cross-linked elastomericcompound. Any elastomeric material which meets all the purity andphysical requirements listed above may serve. Most preferably, Sarlink™2450 elastomeric material from DSM having a hardness of about 50 on theShore A scale is used to form gasket 26.

Sensor pad 30, also shown in FIGS. 6 and 7, can be formed of a materialsimilar to that used to form gasket 26. Pad 30 is formed of a durableorganic polymer which does not creep or flow when stressed, and has alow durometer. Preferably, a material used to form pad 30 has a hardnessof between 40 and 60 on the Shore A scale. Most preferably, a siliconerubber or material such as Sarlink 2450 is used to form pad 30.

According to the present invention, a sample chamber 54 of any size canbe fabricated. Fabrication of a large sample chamber may be advantageousin some circumstances. As noted above, however, in the field ofelectrochemical analysis of blood, it is commonly desirable to performas many analyte analyses as possible on a very small volume of blood.Thus, according to a preferred embodiment of the present invention, itis desirable to fabricate sensor 28 with a sample chamber 54 that is assmall as possible. Using the novel materials and methods of the presentinvention a sensor may effectively be utilized for a period of at leastthirty (30) days, or the measurement of least one thousand (1,000) bloodsamples having a sample chamber with a volume of less than about 10.0 μl(microliters); and preferably, from about 3.0 to about 5.0 μl.

Referring now to FIGS. 9A and 9B through 11, 25, and Table I, a planarelectrochemical sensor 28 in accordance with a preferred embodiment ofthe present invention is shown. FIG. 9A also shows phantom outlines ofsample chamber 54, inlet 56 and outlet 58. These features are shown toillustrate the relative position of electrodes 86, 88, 90, and 92described below to the flow path of a sample to be tested. Sensor 28includes substantially planar substrate 62, conductive metal strips 64,66, 68, and 70 deposited thereupon, and dielectric layer 72 deposited onsubstrate 62 so as to cover portions of conductive strips 64, 66, 68,and 70, while leaving portions of some of the strips uncovered.

Substrate 62 is formed from any substantially electrically insulatingmaterial such as ceramic, glass, refractory, polymers or combinationsthereof. Formation of such an insulating substrate as a mechanicalsupport or base is common knowledge to those of ordinary skill in theart. In the preferred embodiment, the substrate comprises approximately96% alumina and approximately 4% glass binder. A suitable materialcomprising the preferred composition is available from Coors CeramicCompany, Grand Junction, Colo. Although in the preferred embodiments ofthe present invention a single substrate forms the foundation of sensor28, a plurality of substrates can also be used, each supporting separatesensor components, and/or helping to support sensor components supportedby other substrates.

Conductive strips 64, 66, 68 and 70 are deposited atop substrate 62 soas to extend from a first end 74 to a second end 76 thereof in apreferred embodiment. At first end 74, the conductive strips aretypically deposited so as to be wide enough to define contact pads 78,80, 82, and 84, respectively. At second end 76, the conductive stripsare typically deposited so as to be somewhat narrower, exposed regionsof which may define electrodes, as described below.

Conductive strips 64, 66, 68 and 70 may be deposited using well knownthin or thick-film techniques. Typically, a compound including a metalis applied via typical thick-film screening to substrate 62, and theapplied compound and substrate are then fired to sinter the active metaland to co-adhere the active metal to the substrate. The electroactivemetal may comprise any conductive metal, for example, silver, platinumor gold, which is not oxidized or reduced in a potential range in whichoxidation or reduction of any species to be measured occurs.Additionally, materials selected for fabrication of conductive strips64, 66, 68 and 70 are desirably selected so as to be free of anyimpurities such as battery metals (electrochemically active in water)which are typically present in off-the-shelf materials commerciallyavailable for wire bonding, soldering, or welding. See EP-A-9481090.2 orU.S. Ser. No. 08/045,847 filed Apr. 9, 1993 which is incorporated hereinby reference.

Many thick-film pastes suitable for use in the present invention arecommercially available, such as a silver pastes available as productnumber 3571UF/Ag from Metech, Inc., of Elverson, Pa. (Metech), silverchloride available as product number 2539/Ag/AgC1 from Metech; goldpastes available as product number PC 10231/Au from Metech, and platinumpaste available as product number PC10208/Pt from Metech.

With specific regard to conductive strip 66, which defines in part aworking electrode 90 a preferred material is a very high purity platinumthick-film paste. Conductive strip 68 preferably comprises a layer ofsilver deposited atop substrate 62 with a layer of silver/silverchloride deposited thereupon in the electrode region, discussed below,to create a reference electrode 86. A layer of cellulose acetate isdeposited atop the layer of silver chloride. Conductive strips 64, 66and 70 comprise a platinum thick-film paste in preferred embodiments.

Employment of a silver reference electrode is within the scope of thepresent invention. Modification of the teachings of the presentinvention with respect to voltage settings, upon the substitution of asilver reference electrode for a silver/silver chloride referenceelectrode, would be easily made by one of ordinary skill in the art.

At the second end 76 of substrate 62, dielectric layer 72 is depositedso as to cover portions of conductive strips 64, 66, 68 and 70, whileleaving portions of the conductive strips uncovered so as to definereference electrode 86, counter electrode 88, working electrode 90,interference correcting electrode 92, and contact pads 78, 80, 82, and84. Material selected for fabrication of the dielectric layer 72 isdesirably electrically insulating and non-porous, free of impuritieswhich may be subject to oxidation or reduction in the potential range ofany species or analyte to be measured, as described above, and isfurther selected so as to be free of mobile ions that would potentiallycarry charge and interfere with the activity of any electrolyte employedin the sensor. Further, dielectric 72 is selected so as to firmly adhereto substrate 62 and conductive strips 64, 66, 68, and 70, so as to allowelectrodes 86, 88, 90, and 92 to be electrically addressable, whileeffectively electrically insulating portions covered by the dielectric.Materials such as ceramics, glass, refractory materials, polymericmaterials, or combinations thereof are well known as dielectricmaterials and are suitable for use as a dielectric in the presentinvention. A preferred material is commercially available as ProductNumber 9615, a ceramic material from E. I. DuPont de Nemours & Co.,Electronics Department, Wilmington, Del.

With respect to materials advantageously selected for fabrication ofconductive strips 64, 66, 68, and 70, it is noted that materialselection becomes less important in regions of the strips which definecontact pads 78, 80, 82 and 84 and which connect the bonding pads toregions which define electrodes. For example, the contact pads andregions of the conductive strips connecting them to the electrodes maybe fabricated from any conducting material that adheres to substrate 62and that does not interfere with the electrical insulation function ofdielectric layer 72. According to one embodiment, the contact pads andregions of the conductive strips connecting them to the electrodes arefabricated from a gold paste.

In addition to the material selection parameters discussed above, and asdiscussed with respect to selection of the dielectric material, it isadvantageous in the fabrication of a sensor to select materials forfabrication of the substrate, the conductive strips, and the dielectriclayer such that good adherence is achieved between adjacent layers, thatis, delamination is minimized. See EP-A-94810190.2 or U.S. Ser. No.08/045,847 filed Apr. 9, 1993. If good adherence is not achieved,reference, counter, working and interference correcting electrodes 86,88, 90, and 92, will not be well-defined which in one embodiment isdefined by a screen used in the thick-film deposition process, anddisadvantageous electrochemistry will result.

A cross-sectional side view of working electrode 90, taken along sectionline 10--10, is illustrated in FIG. 10. As described above, conductivestrip 66 is deposited upon substrate 62, and dielectric layer 72 coversportions of conductive strip 66 leaving a portion uncovered to define aworking electrode area. An active layer 96, comprising a catalyticallyactive quantity of an enzyme immobilized onto platinized carbon powderparticles, is deposited upon conductive strip 66 using techniquessimilar to the deposition of conductive strips 64, 66, 68 and 70.Typically, thick-film screen printing at low temperature is used toapply an active paste to conductive strip 66 in order to limit thermaldamage to the enzyme, see Table I.

As noted, active layer 96 comprises an enzyme immobilized into anelectrically conducting support member which consists of or comprises aporous layer of resin-bonded carbon or graphite particles. The particleshave intimately mixed therewith, or deposited or adsorbed onto thesurface of the individual particles prior to bonding to form the layer,a finely divided platinum group metal to form a porous, substrate layeronto which the enzyme is adsorbed or immobilized and comprising asubstantially heterogeneous layer of resin-bonded carbon or graphiteparticles with the platinum group metal adsorbed on the carbon orgraphite particles. An enzyme immobilized or adsorbed onto a porouslayer of resin bonded platinized carbon particles is disclosed byMullen, in U.S. Pat. No. 5,160,418 and Bennetto et al., in U.S. Pat. No.4,970,145, both of which are incorporated by reference. The active layer96 may alternatively be formed by first depositing the finely dividedplatinum group metal, optionally preadsorbed onto or admixed with finelydivided carbon or graphite, with or without all or some of the resinbinder, if used, on the surface of the electrically conductivesubstrate, or conductive strip 66.

The platinum group metal in finely divided elemental form, includingplatinum, palladium, iridium, or rhodium, may be replaced by thecorresponding oxides, such as platinum or palladium oxide. Therefore,all references herein to a platinized material are to be taken asincluding a platinum group metal, as described above, and/orcorresponding oxides-containing material unless the context requiresotherwise.

Any suitable carbon or graphite powder which readily permits thesubsequent immobilization of an enzyme may be used to form the activelayer. To this end, carbon powder should be used having a high densityof functional groups, such as carboxylate, amino and sulfur-containinggroups, on the surface, as opposed to the more vitreous and glassycarbons, which bind enzymes only poorly. Typically, carbon or graphitepowder particle size ranges from between about 3.0 and about 50.0 nm;preferably, particle sizes range from between about 5.0 and 30.0 nm.

Platinum may be deposited on the carbon particles in any convenientfashion, for example, vapor phase deposition, electrochemicaldeposition, or simple adsorption from colloidal suspension to giveplatinum group metal loadings in the range of between about 0.1 to about20.0 percent, by weight, based on the weight of carbon. Preferably, theplatinum group metal loadings are between about 5.0 to about 15.0percent by weight. These limits are, however, practical rather thancritical. Below about 1.0 percent platinum group metal, the outputsignal falls to a level which, in practical terms, is too low to bemeasured except by very sensitive apparatus; above about 20.0 percent,the loading of platinum group metal becomes uneconomic, with littleadditional benefit in terms of increased response or sensitivity. In thepreferred technique, the carbon powder is platinized by the oxidativedecomposition of a platinum compound such as chloroplatinic acid or,more preferably, a complex of platinum or palladium with an oxidadizableligand, in the presence of the carbon powder, thereby to depositcolloidal size platinum or palladium direct upon the surface of thecarbon particle, in the manner taught, for example, by Petrow et al., inU.S. Pat. Nos. 4,044,193 and 4,166,143, both of which are incorporatedherein by reference. Preferably, the platinum group metal or oxideparticles have a particle size in the range of between about 1.0 nm toabout 20.0 nm, and most preferably are of a colloidal size in the rangeof between about 1.0 nm to about 4.0 nm.

The preferred active layer substrate used in accordance with the presentinvention are, in fact, commercially available materials, sold under thename PLATINUM ON CARBON BLACK from E-TEK, Inc., Framingham, Mass. Anenzyme, such as glucose oxidase, or lactate oxidase, can be immobilizedonto platinized carbon powder particles, prepared by the deposition ofcolloidal platinum having a particle size of between about 1.5 to about2.5 nm onto the carbon powder, having a nominal particle size of about30.0 nm, by the oxidative decomposition of complex platinum sulfite acid(II) using H₂ O₂.

In the present invention, the platinum activated carbon is treated in aphosphate buffer formulation having a pH of about 7.5. The platinumactivated carbon is added to the buffer to neutralize any sulfuric acidpresent from the formation of the platinized carbon powder particles. Tothe platinum activated carbon and buffer mixture a co-protein, such asbovine serum albumin, is added to adsorb onto the carbon. The bovineserum albumin is added to help stabilize the enzyme, such as glucoseoxidase, as is known to those skilled in the art. A binder, such as acommercially available resin solution sold under product number 8101RSfrom Metech, is then added to the bovine serum albumin-platinumactivated carbon mixture. The binder material, as noted above, acts tohold the components of the active layer together. To this mixture, asurfactant may be added to provide better printing flow characteristicswhen active layer 96 is screen printed upon conductive strip 66. Anadditional benefit of the surfactant is to act as a wetting agent forthe sensor during use. The active layer 96 being comprised of ahydrophobic binder becomes difficult to wet with water after it is fullydried. The surfactant facilitates this wetup. The surfactant materialused can be any liquid surfactant, known to those skilled in the art,which is water soluble and exhibits a hydrophilic lipophilic balance(HLB) in the range of 12-16. Typical surfactant materials for use inthis regard can be alkylarylpolyether alcohols, such asalkylphenoxypolyethoxyethanol. One such material is sold under thetrademark Triton® from Union Carbide Chemicals and Plastics Co., Inc.,Danbury, Conn. The preferred material for use in the present applicationis Triton® X-100 surfactant (HLB 13.5). After these components aremilled, a resin thinner may be added to adjust the active layer 96viscosity for printing purposes. Typically, a petroleum solvent-basedresin thinner is used to bring the paste viscosity within the range ofbetween 10,000 to about 100,000 centipoise. Resin thinners for thispurpose are commercially available as product number 8101 thinner fromMetech. An enzyme, such as glucose oxidase or lactate oxidase, is thenadded to the mixture, and the final paste is screen printed uponconductive strip 66. Other enzymes may be similarly added to the mixtureto prepare active layers specific for other analytes.

It is preferred to put the active layer down last, i.e. beforedepositing the cover membrane, to minimize the thermal impact to theenzyme from other steps in the sensor formation, see Table I and FIG.25.

Interference correcting electrode 92 is formed in a manner similar tothe working electrode 90. The interference correcting electrode 92,however, includes an inactive layer (not shown) which is made using thesame components and method used in a process of forming the workingelectrode, however, an inactive or nonreactive protein, such as thebovine serum albumin is added to the mixture of bovine serumalbumin-platinum activated carbon, resin, surfactant, and thinner. Asnoted above, the interference correcting electrode serves to adjust forany interfering species, such as the neutral species acetaminophen,diffusing through a semi-permeable membrane layer 94 (discussed below)on top of electrodes 86, 88, 90, and 92.

Referring now to FIG. 11, a cross-sectional side view of referenceelectrode 86, taken along section line 11--11, is shown. Referenceelectrode 86, as noted above, is formed as a conductive strip 68,preferably comprising a layer of silver is deposited thereon. Dielectriclayer 72 is deposited covering a portion of conductive strip 68, whileleaving a portion uncovered to define the electrode areas and contactpads. A silver/silver chloride layer 102 is deposited upon conductivestrip 68 by screen printing techniques known to those of skill in theart. Silver/silver chloride reference electrode inks, such as thoseavailable as product number 2359 from Metech, are developed to provide astandard reference electrode utilizing the silver/silver chloridecouple.

Reference electrode stability measurements showed that over a period ofseveral days, the potential of the reference electrode (86) shifted uponexposure of the sensor to whole blood. The root cause of the problem wasidentified as a gradual decrease in the rejection properties of themembrane (94) allowing penetration by blood proteins, which fouled thereference. Cellulose acetate was chosen as a shield for the referenceelectrode due to its barrier properties to proteins and its ability totransport sufficient water and electrolytes to maintain a stablepotential at the surface of the printed silver/silver chloride.

The choice of a proper solvent and cure process is critical in preparinga uniform cellulose acetate layer over the reference electrode. Thesolvent must have a low vapor pressure (high boiling point) in order toprovide sufficient screen life for the printing process to be completed.It must be compatible with the printing screens, i.e. not degrade thescreen emulsion during priming. The viscosity of the prepared paste mustbe relatively high, 40,000 centipoise to 350,000 centipoise. Thismandates that the % solids of the polymer solution be fairly high, thusthe solvent must be very good for the polymer. Suitable solvents includethe so-called "super solvents", polar aprotic solvents such as dimethylformamide, dimethylsulfoxide, hexamethylphosphoramide, and1,3-dimethyl-2-imidazolidinone (DMI) are examples of this class ofsolvent. One final restriction was that the solvent not be a carcinogen,mutagen, or teratogen in order that it might be handled more readily bythe formulation technician and the screen printer. The preferred solventis DMI.

Solutions prepared in the concentration range of from 15 to 35 grams ofcellulose acetate in 100 mL of DMI were found acceptable for theprinting process. The preferred concentration was chosen as 20 gramscellulose acetate in 100 mL of DMI. In order to rapidly dissolve thepolymer, the solvent is heated to between 60° and 100° C., with thepreferred temperature being 95° C. The polymer is added to the rapidlystirred (magnetic stir bar), heated solvent (water bath with thetemperature preset). It is then mechanically mixed in with a spatula,after which it is stirred continuously until completely dissolved. Thepolymer/solvent mixture (paste) is then removed from the water bath,allowed to cool to room temperature, labeled and set aside until neededfor printing.

The paste is generally printed on the same day it is prepared, it can beused up to several months after preparation, however, performance of thelayer gradually decreased with paste shelf life. The paste is applied ina 2 pass print after which it is allowed to level for a period of timeno less than 10 minutes and no more than one hour. It is cured in a boxoven at 55° C. for 10 minutes, the temperature of the oven is thenramped up to 100° C. over a 10 minute period, the curing continues for10 minutes more at this temperature. This print method/cure cycle iscrucial to the performance of the cellulose acetate membrane. Low curetemperatures do not remove sufficient solvent, while longer cures orhigher cure temperatures lead to a brittle membrane which delaminateseasily from the substrate, particularly after post-treatment of thesensor with an anti-drying agent. Printing with more passes leads to athicker membrane, which is also prone to delamination. It is importantnot to completely remove solvent, as complete removal would hinder thehydration process.

A layer of cellulose acetate 100 is applied over the silver/silverchloride layer 102 to protect the silver chloride from contaminantspresent in blood samples that would shift the reference potential. Thecellulose acetate layer 100 can be applied by a spotting technique or bya screen printing technique. If the spotting technique is used, anAsymtek XYZ table, available from Asymtek Corporation, Carlsbad, Calif.,and known to those of skill in the art, will be used. If a screenprinting deposition process is used, a high viscosity solution from ahigh boiling solvent, such as 2-(2-ethoxyethoxy)ethylene acetate will beused.

Lastly, as noted above, each electrode 86, 88, 90 and 92 is covered witha glucose and oxygen-permeable membrane 94.

Membrane 94 can be formed from cellulose acetate, polyurethane, siliconecompounds, and other membrane materials known to those skilled in theart such as Nafion® material available from E. I. DuPont de Nemours,Wilmington, Del. The preferred membrane 94 is a dispersion of apolymerizable silicon-containing compound applied in an incompletelycured form of a silicone compound dispersed phase in a liquid carrier.The carrier is essentially insoluble in the dispersed phase andremovable from the dispersion during curing. The dispersion will dry andcure as a continuous layer, film or membrane, having a high glucose andoxygen permeability to function as a single membrane in anelectrochemical glucose sensor. A single-layered, semi-permeablemembrane is disclosed by Jones, in EP Pat. No. 207 370 B1 which isincorporated herein by reference. The silicon-containing compound may bedispersed in the continuous phase as an oligomer, prepolymer, orincompletely cured polymer.

The polymerizable silicon-containing compound, after dispersion in acontinuous phase, such as by including an emulsifier, can be cured inany known manner during removal of the continuous phase, such as byevaporation of water from a water-continuous phase silicone emulsion ordispersion, as disclosed by Johnson et al., in U.S. Pat. No. 4,221,688,and Elias, in U.S. Pat. No. 4,427,811, both of which are incorporatedherein by reference. Further, the dispersion of the silicon-containingcompound can include a suitable curing catalyst, or can be heat cured,so the dispersion of the polymerizable silicon-containing compound isapplied as a layer in the form of an incompletely cured dispersion andat least a portion of the carrier or continuous phase is removed fromthe dispersion during final curing. The emulsion can consist of adispersion of silicone latex particles and silica. Upon evaporation ofwater, the silicone latex particles are cross-linked by the silica. Themorphology of the resulting membrane is polydiorgano cross-linkedparticles bounded by a continuum of silica or silicates. It is thesilica phase in which analyte transport, i.e. glucose, lactate, etc.,takes place.

In accordance with one aspect of the present invention, thepolymerizable silicon-containing compound is an organosiloxane, andparticularly a diorganosiloxane, comprising essentially a linear speciesof repeating diorganosiloxane units which may include small numbers ofmonoorganosiloxane units up to a maximum of about one unit for each 100diorganosiloxane units wherein the polymer chain is terminated at eachend with silicone-bonded hydroxyls.

In accordance with another important aspect of the present invention,the polymerizable silicone-containing compound forming an oxygen andglucose-permeable membrane is applied onto an electrode as an aqueoussilicone emulsion comprising a continuous water phase and ananiionically stabilized dispersed silicone phase wherein the siliconephase is a graft copolymer of a water soluble silicate and a hydroxylendblocked polydiorganosiloxane. As disclosed by Saam, in U.S. Pat. No.4,244,849, incorporated herein by reference, such silicone emulsions,having a pH within the range of from about 8.5 to about 12.0, are stableupon extended storage and result in a cured elastomeric continuous layerupon removal of water under ambient conditions. These silicone compoundsare obtained from the interaction of hydroxyl endblockedpolydiorganosiloxanes and alkali metal silicates to form graft polymersanionically stabilized in aqueous emulsions at pH of, for example, 8.5to 12.0. If stability is not important, however, the pH is not critical.The emulsion can be applied in layer form to manufacture the membrane assoon as the components are homogeneously dispersed.

The expression "hydroxyl endblocked polydiorganosiloxane" is understoodto describe an essentially linear polymer of repeating diorganosiloxaneunits containing no more than small impurities of monoorganosiloxaneunits. The hydroxyl endblocked diorganosiloxane will therefore haveessentially two silicon-bonded hydroxyl radicals per molecule. To impartelastomeric properties to the product obtained after removal of thewater from the emulsion, the polysiloxane should have a weight averagemolecular weight (M_(w)) of at least 5,000. Polysiloxanes with weightaverage molecular weights below about 5,000 down to about 90, also areuseful if the polymers form a continuous film or layer upon curing.Tensile strengths and elongations at break improve with increasingmolecular weight, with relatively high tensile strengths and elongationsobtained above 50,000 M_(w). However, since in a preferred embodiment ofthe invention, the cured polymers are bonded directly to an electrode,and do not undergo any severe mechanical stress during use, highstrength is not necessary for the polymer to be useful. The maximumM_(w) is one which can be emulsified or otherwise dispersed in a liquidcarrier or continuous phase, such as water. Weight average molecularweights up to about 1,000,000 for the incompletely cured dispersedpolysiloxane are practical for use in the sensor of the presentinvention. Upon curing, there is no upper limit to the molecular weightof the membrane. The preferred M_(w) for the polymerizable dispersedsiloxane is in the range of 1,000 to 700,000.

Organic radicals on useful hydroxyl endblocked polydiorganosiloxanes canbe, for example, monovalent hydrocarbon radicals containing less thanseven carbon atoms per radical and 2-(perfluoroalkyl)ethyl radicalscontaining less than seven carbon atoms per radical. Examples ofmonovalent hydrocarbon radicals include methyl, ethyl, propyl, butyl,isopropyl, pentyl, hexyl, vinyl, cyclohexyl and phenyl; and examples of2-(perfluoroalkyl)ethyl radicals include 3,3,3-trifluoropropyl and2-(perfluorobutylmethyl). The hydroxyl endblocked polydiorganosiloxanespreferably contain organic radicals in which at least 50 percent aremethyl. The preferred polydiorganosiloxanes are the hydroxyl endblockedpolydimethylsiloxanes.

In accordance with one important aspect of the present invention, thehydroxyl endblocked polydiorganosiloxane is employed as an anionicallystabilized aqueous emulsion. For the purposes of this embodiment"anionically stabilized" means the polydiorganosiloxane is stabilized inemulsion with an anionic surfactant. The most preferred anionicallystabilized aqueous emulsion of hydroxyl endblocked polydiorganosiloxaneare those prepared by the method of anionic emulsion polymerizationdescribed by Findlay et al., in U.S. Pat. No. 3,294,725, herebyincorporated herein by reference. Another method of preparing hydroxylendblocked polydiorganosiloxanes is described by Hyde et al., in U.S.Pat. No. 2,891,920, also incorporated herein by reference.

An alkali metal silicate or colloidal silica must be included in theemulsified silicone composition for the preparation of extended storagestable emulsions used in the invention. The alkali metal silicatespreferred for use in the emulsions forming the oxygen and low molecularweight analyte-permeable membranes of the present invention are watersoluble silicates. The alkali metal silicate is preferably employed asan aqueous solution. Aqueous silicate solutions of any of the alkalimetals can be employed, such as lithium silicate, sodium silicate,potassium silicate, rubidium silicate and cesium silicate.

The colloidal silicas are well known in the art and commerciallyavailable, and can be included in the dispersion for increased strengthand storage stability. Although any of the colloidal silicas can beused, including fumed and precipitated colloidal silicas, silicas in anaqueous medium are preferred. Colloidal silicas in an aqueous medium areusually available in a stabilized form, such as those stabilized withsodium ion, ammonia or an aluminum ion. Aqueous colloidal silicas whichhave been stabilized with sodium ion are particularly useful for formingan emulsion because the pH requirement can be met without having to addother components to bring the pH within the range of, for example, 8.5to 12.0. The expression "colloidal silica" as used herein are thosesilicas which have particle diameters of from about 0.0001 to about 0.1micrometers. Preferably, the particle diameters of the colloidal silicasare from about 0.03 to about 0.08 micrometers; most preferably, thesilica particle diameter is about 0.06 micrometers.

The colloidal silica can be added to the anionically stabilizedhydroxylated polydiorganosiloxane in the form of a dry powder or as anaqueous dispersion. Preferably, the colloidal silica is added in theform of a sodium ion stabilized aqueous dispersion of colloidal silica,many of which are commercially available. These commercial colloidalsilicas are usually available in aqueous dispersions having betweenabout 10.0 to about 30.0 percent, by weight, colloidal silica, and a pHbetween about 8.5 to about 10.5.

Aqueous solutions of sodium or potassium silicate are well known and arecommercially available. The solutions generally do not contain anysignificant amount of discrete particles of amorphous silica and arecommonly referred to as water glass. The ratio, by weight, of silica toalkali metal oxide in the aqueous solutions of alkali metal silicates isnot critical and can be varied between about 1.5 to about 3.5 for thesodium silicates, and about 2.1 to about 2.5 for the potassiumsilicates. The aqueous alkali metal silicate solutions are particularlyuseful in preparing the emulsions used in the present invention becausethe addition of the silicate solution often brings the pH of theemulsion within the range of about 8.5 to about 12.0 so that additionalingredients are not necessary to adjust the pH of the emulsion. Ofcourse, other aqueous alkali metal silicate solutions, such as thoseprepared by hydrolyzing silicon esters in aqueous alkali metal hydroxidesolutions, can also be employed in the present invention.

In accordance with one aspect of the present invention, thepolymerizable silicon-containing compound is dispersed by combining anaqueous solution of an alkali metal silicate and the polymerizablesilicon-containing compound in an emulsion so that a graft copolymer isformed as dispersed particles. The preferred procedure for preparingsilicone emulsions is to add the alkali metal silicate to an anionicallystabilized aqueous emulsion of one or more hydroxyl endblockedpolydiorganosiloxanes, adjust the pH of the emulsion within the range ofabout 8.5 to about 12.0, and then age the emulsion for a period to forman elastomeric product upon removal of the water under ambientconditions. In this procedure, the pH of the emulsion containingdissolved silicate and dispersed hydroxyl endblockedpolydiorganosiloxane is important to the formation of the emulsion. A pHof 8.5 to 12.0 maintains the alkali metal silicate dissolved so thatsufficient graft copolymerization between the dissolved silicate anddispersed siloxane occurs during removal of the carrier (e.g., water) toproduce an emulsion capable of providing polymerization, or furtherpolymerization, of the silicon-containing compound when deposited as alayer to form a membrane. If the pH is lower than the stated range,silicic acid is formed from the alkali metal silicate. Silicic acid isunstable and rapidly polymerizes by condensation, which can gel theemulsion. Since silicic acid formation is almost completely suppressedat a pH of between about 10.0 to about 12.0, and the reaction betweendissolved alkali metal silicate and dispersed siloxanes occurs morerapidly within this pH range, this range is preferred for emulsionscontaining an alkali metal silicate.

Silicone emulsions prepared by silicate copolymerization are aged at apH range of between about 8.5 to about 12.0 for a period sufficient toallow interaction between the dissolved silicate and the dispersedsiloxane so that an elastomeric product is formed upon removal of thewater under ambient conditions. The aging period is effectively reducedwhen an organic tin salt is employed in an amount between about 0.1 toabout 2.0 parts, by weight, of polydiorganosiloxane. The organic tinsalts expected to be useful in the emulsions include mono-, di andtriorganotin salts. The anion of the tin salt employed is not criticaland can be either organic or inorganic, although organic anions such ascarboxylates are generally preferred. Organic tin salts that can beemployed include octyltin triacetate, dioctyltin dioctoate, didecyltindiacetate, dibutyltin diacetate, dibutyltin dibromide, dioctyltindilaurate and trioctyltin acetate. The preferred diorganotindicarboxylate is dioctyltin dilaurate.

The relative mounts of alkali metal silicates and hydroxyl endblockedpolydiorganosiloxane employed can vary over a considerable range.Preferred elastomer properties are obtained when between about 0.3 toabout 30 parts, by weight, silicate is employed for each 100 parts, byweight, siloxane.

In accordance with one aspect of the invention, an alkyl tin salt isadded to the dispersion to catalyze the curing of the final emulsionduring the devolatization, or other removal, of the carrier to yield thecured membrane. Preferred salts are dialkyltin dicarboxylates such asdibutyltin diacetate, dibutyltin dilaurate, and dioctyltin dilaurate;the most preferred tin salt is dibutyltin dilaurate. The emulsion ofcatalyst is used in an amount sufficient to yield between about 0.1 toabout 2.0 parts, by weight, of the alkyl tin salt for each 100 parts, byweight, of the polymerizable silicon-containing compound, such aspolydiorganosiloxane. Larger amounts could by used, but would serve nouseful purpose.

The dispersion of the polymerizable silicon-containing compound(s) cancontain components in a broad range of concentrations. The preferredconcentrations will depend on the thickness of the membrane desired. Forexample, to provide a thin elastomeric membrane (20 microns) that doesnot form cracks as the carrier or continuous phase evaporates, it isbest to use a dispersion having a combined amount of silicate andpolydiorganosiloxane in the range of between about 67.0 to about 160.0parts, by weight, for each 100 parts, by weight, of carrier such aswater. Preferred membrane thicknesses are between about 10.0 to about100.0 microns, preferably about 20.0 microns.

If an emulsifying agent is incorporated into the composition to form thedispersion the amount of emulsifying agent can be less than about 2.0percent, by weight, of the emulsion. The emulsifying agent can resultfrom neutralized sulfonic acid used in the emulsion polymerizationmethod for the preparation of a hydroxyl endblockedpolydiorganosiloxane.

Anionic surfactants are preferably the salts of the surface activesulfonic acids used in the emulsion polymerization to form the hydroxylendblocked polydiorganosiloxane. The alkali metal salts of the sulfonicacids are preferred, particularly the sodium salts. The sulfonic acidcan be illustrated by aliphatically substituted benzenesulfonic acids,naphthalene sulfonic acids, and diphenylether sulfonic acids, aliphaticsulfonic acids, and silylalkylsulfonic acids. Other anionic emulsifyingagents can be used, for example, alkali metal sulforicinoleates,sulfonated glyceryl esters of fatty acids, salts of sulfonatedmonovalent alcohol esters, amides of amino sulfonic acid such as thesodium salt of oleyl methyltauride, sulfonated aromatic hydrocarbonalkali salts such as sodium alpha-naphthalene monosulfonate,condensation products of naphthalene sulfonic acids with formaldehyde,and sulfates such as ammonium lauryl sulfate, triethanol amine laurylsulfate and sodium lauryl ether sulfate.

Nonionic emulsifying agents can also be included in the emulsion (inaddition to the anionic emulsifying agents). Such nonionic emulsifyingagents are, for example, saponins, condensation products of fatty acidswith ethylene oxide such as dodecyl ether of tetraethylene oxide,condensation products of ethylene oxide and sorbitan trioleate,condensation products of phenolic compounds having side chains withethylene oxide, such as condensation products of ethylene oxide withisododecylphenol, and imine derivatives such as polymerized ethyleneimine.

The polymerizable silicon-compound dispersion used to form the oxygenand glucose-permeable membranes of the present invention may containadditional ingredients to modify the properties of the dispersions, orthe cured polymeric membrane products obtained from the dispersions. Forexample, a thickener may be added to modify viscosity of the dispersionor to provide thixotropy for the dispersion. An antifoam agent may beadded to the dispersion to reduce foaming during preparation, coating orcuring in layer form.

Fillers may be added to the dispersion to reinforce, extend or pigmentthe membrane. Useful fillers include colloidal silica, carbon black,clay, alumina, calcium carbonate, quartz, zinc oxide, mica, titaniumdioxide and others well known in the art. These fillers should be finelydivided and it may be advantageous to use aqueous dispersions of suchfillers.

The filler preferably has an average particle diameter of less thanabout 10.0 micrometers. When the silicone emulsions are spread out forfinal curing to form the oxygen and glucose-permeable membranes of thepresent invention, the water, or other nonsolvent carrier, evaporates,or is otherwise removed, to leave a cured oxygen and glucose-permeablemembrane. Evaporation of the carrier is usually complete within a fewhours to about one day depending on the dispersion film thickness andmethod of application. Another of the important advantages of thepresent membrane is excellent adhesion to both polar and nonpolarsubstrates.

One of the more important advantages of the oxygen and analyte-permeablemembranes used with the present invention, is the capability of thesemembranes to be bonded to an electrode activated with a suitable enzymecatalyst, such as glucose oxidase, glucose dehydrogenase or lactateoxidase. In accordance with one embodiment of the present invention, acompound capable of catalyzing the reaction of glucose with oxygen isincorporated within the anode, or active layer 96, and the oxygen andglucose-permeable membrane 94 of the present invention is coated overthe reference, counter, working (including active layer 96), andinterference correcting electrodes 86, 88, 90, and 92.

The membrane materials described herein are very compatible with wholeblood 98, have a durable surface and are highly selective to oxygenpenetration so that a sufficient stoichiometric excess of oxygenpermeates the membrane 94 even from whole blood.

The preferred materials for membrane 94 are an anionically stabilized,water-based hydroxyl endblocked polydimethylsiloxane elastomercontaining at least about 10.0 percent silica, by weight. Mostpreferably, the elastomer contains about 14.0 percent, by weight,colloidal silica, and is commercially available as FC-61 coating fromDow Corning, Midland, Mich. This material is a low viscosity, filled,opaque emulsion. Typically, this material has a pH of about 11.0, and aviscosity of about 40,000 cp.

In another aspect of the present invention, it has been found thatduring dry storage of sensors 28, membranes 94 become increasingly moredifficult to wetup. It is believed that residual water and othersolvents, initially present in the membrane 94 after casting, evaporateduring storage and cause coalescence of silicone agglomerates. Thistightening of the membrane structure can decrease the sensitivity andincrease the response time of the sensor toward glucose.

The sensors 28 can be post-treated to prevent the membrane from agingduring dry storage, for example, by preventing the membranes from fullydrying with humidification, or treatment with a high boiling point,water soluble, hydrophilic polymer liquid antidrying agent, such assurfactants or polyethylene glycols. Preferably, a non-ionic surfactant,having a molecular weight of at least about 300, such as Triton® X-100surfactant, Tergitol® 15 surfactant from Union Carbide Chemicals andPlastics Co., Inc., Danbury, Conn, Tween® 20 ethoxylated sorbitan esterssurfactant from ICI Surfactants, Wilmington, Del., and polyethyleneglycols having molecular weights between about 200 and 600, are appliedfor post-treatment of sensors 28 to improve output, and response time,while minimizing sensor drift, upon initial start-up. The preferredmaterial for post-treatment is polyethylene glycol having a molecularweight of about 400.

Referring again to FIGS. 9A and 9B, while noting that a variety ofsensor configurations can be advantageous in different applications, thefollowing non-limiting preferred dimensional specifications of a sensor28 fabricated in accordance with a preferred embodiment of the presentinvention are given.

Substrate 62 can be fabricated in a variety of shapes and sizes.According to one specific preferred embodiment of the invention,substrate 62 is from about 0.4 inch to about 0.5 inch long; preferably,about 0.45 inch long. Substrate 62 is from about 0.15 inch to about 0.25inch wide; preferably, about 0.18 inch wide. Substrate 62 is from about0.02 inch to about 0.05 inch thick; preferably, about 0.025 inch thick.Conductive strips 64, 66, 68 and 70 are each deposited in a thickness offrom about 10.0 microns to about 20.0 microns; preferably, the stripsare about 15.0 microns thick. Conductive strips 64, 66, 68, and 70, atend 76 of the sensor, are from about 0.01 inch to about 0.03 inch wide,preferably about 0.01 wide. Contact pads 78, 80, 82, and 84 at end 74 ofthe sensor, are from about 0.025 inch to about 0.05 inch wide,preferably about 0.03 inch wide.

Dielectric layer 72 is preferably deposited in a thickness of from about10.0 microns to about 50.0 microns, preferably about 20.0 microns thick.Thickness values are given after firing or curing.

Portions of the conductive strips are exposed to define the referenceelectrode 86, counter electrode 88, working electrode 90, andinterference correcting electrode 92. The exposed surface area for thereference electrode 86 is about 0.00015 inch², and for the counterelectrode 88 is about 0.00022 inch². The working and interferencecorrecting electrodes 90, 92, each have surface areas of about 0.00038inch². These exposed surface area dimensional specifications do not takeinto consideration surface area due to the edges of the electrodes,defined by the thickness of the electrodes as deposited or the porosityof the layer. Such edge dimensions are minimal relative to the overallelectrode areas. However, the exposed surface area specification arethus somewhat approximate.

A cellulose acetate layer 100 is applied over the silver/silver chloridelayer 102 of the reference electrode 86, which was deposited over theexposed portion of conductive strip 68. The cellulose acetate layer 100protects the silver chloride from contaminates that would shift thereference potential.

The active and inactive layers are then applied over the exposedportions of conductive strips 66 and 70, forming the working andinterference correcting electrodes 90, 92, respectively.

Cover membrane 94 is then deposited, preferably spun-cast, to a totalthickness from about 5.0 microns to about 50.0 microns, preferably fromabout 10.0 microns to about 20.0 microns. The cover or protectivemembrane 94 is preferably applied in layers to enable thin overallthickness with required permeability characteristics.

The present invention will be further illustrated by the followingexamples which are intended to be illustrative in nature and are not tobe construed as limiting the scope of the invention.

EXAMPLE I

Referring to FIGS. 9 and 9B and 25 and Table I, one suitableconstruction of a solid state, planar glucose sensor 28 including thecomponents and design substantially in accordance with an aspect of thepresent invention is provided by the following combination of elements.

A partial assembly of planar glucose sensor 28 having substrate 62 andconductive metal strips 64, 66, 68 and 70 was fabricated in accordancewith a method of the present invention on a 0.025 inch thick, 0.18 inchby 0.45 inch, electrically nonconducting substrate 62 comprisingapproximately 96% alumina and approximately 4% glass binder, availablefrom Coors Ceramic Company, Grand Junction, Colo. Portions of conductivestrips 64, 66, 68 and 70, as well as contact pads 78, 80, 82 and 84,were deposited onto the substrate using a screen printing technique,with a 10.0 micron emulsion of gold conductor paste, available asproduct number PC10231 from Metech, Inc., Elverson, Pa. A stainlesssteel screen having a 325 mesh pattern was used to screen print the goldpaste onto substrate 62. Conductor strips 64, 66 and 70 were connectedwith platinum upper portions, as the conductive strips were continuedtoward second end 76. These strips were fabricated by screen printing a10.0 micron emulsion high purity platinum conductor paste, available asproduct number PC10208 from Metech, onto the substrate. A screen similarto that described above was used to deposit the platinum conductorcomposition. Conductor strip 68 was similarly continued toward secondend 76 by applying a 10.0 micron emulsion silver conductor paste,available as product number 3571UF from Metech, onto the substrate. The325 mesh screen made of stainless steel wire was used to screen printthe silver conductor paste. A 10.0 micron emulsion silver/silverchloride reference electrode ink, available as product number 2539 fromMetech, was subsequently screen printed over a portion of conductivestrip 68 at end 76, covering an area of conductive strip 68 at least aslarge as, and preferably larger than, the area of conductive strip 68 tobe exposed by dielectric layer 72 to define reference electrode 86.Lastly, a cellulose acetate layer 100, available as product number18095-5 from Aldrich Chemical Co., Milwaukee, Wis., was screen printedover the silver/silver chloride reference electrode 86. This layer isapplied over the reference electrode to protect the silver chloride fromcontamination that could shift the reference potential.

In this example a BTU 7 zone furnace with a 3 zone dryer, from Fast Fireof Billerica, Mass., was used in firing the inorganic pastes. Firing wascarried out per the manufacturer's recommendations, ramped to the peakconditions. The gold conductor paste was fired at 850° C. for a 10minute peak, the platinum conductor paste was fired at 750° C. for a 13minute peak, and the silver conductor ink was fired at 750° C. for a 10minute peak.

Conductive strips 64, 66, 68 and 70 were deposited on substrate 62 so asto be 0.01 inch wide at end 76; contact pads 78, 80, 82 and 84 weredeposited on substrate 62 so as to be 0.03 inch wide, and 0.8 inch longat end 74.

A dielectric material 72, available as product number 9615 from DuPontElectronics, Wilmington, Del., was screen printed as a 15.0 micronemulsion over a large portion of sensor 28, extending from second end 76to contact pads 78, 80, 82 and 84. A 325 mesh screen made of stainlesssteel was used for the screen printing process. The dielectric was firedat 750° C. for a 10 minute peak. As noted above, portions of conductivestrips 64, 66, 68 and 70 were not covered by dielectric 72, exposingtheir electrode areas.

Silver/silver chloride is applied at 75° C. for 30 minutes.

Cellulose acetate is applied at 55° C. for 10 minutes, ramped to 100° C.for 10 minutes, and then 10 minutes at 100° C. (30 minute cure time).

An active layer 96, comprising a catalytically active quantity ofglucose oxidase, available from Biozyme Laboratories International,Ltd., San Diego, Calif., immobilized onto platinized carbon powderparticles, available from E-TEK, Inc., Framingham, Mass., was depositedupon conductive strip 66 to form working electrode 90 also using a thickfilm screen printing technique. An inactive layer, comprising aninactive protein, such as bovine serum albumin, sold under the trademarkPentex® bovine albumin from Miles, Inc., Kankakee, Ill., immobilizedonto platinized carbon powder particles, available from E-TEK, wasdeposited upon conductive strip 70 to form interference correctingelectrode 92 using similar thick film screen printing techniques. Thefabrication of the active and inactive layers is described in furtherdetail in Example II.

After the conductive strips, dielectric layer, and electrodes aredeposited onto substrate 62 and the contact are masked to preventelectrode "shunting", a cover membrane 94 is spun-cast over theelectrode area of the sensor. An anionically stabilized, water-basedhydroxyl endblocked polydimethylsiloxane elastomer, comprising about 14percent, by weight, colloidal silica, commercially available as FabricCoating (FC)-61 from Dow Corning, Midland, Mich., was applied to thesensor 28 using a spin-casting technique. An IVEK laboratory pump and anIntegrated Technologies P-6000 spin coater were used to apply the covermembrane 94 in multiple layers over the sensor. The first layer wasapplied by complete flooding of the wafer with the membrane elastomermaterial. The Integrated Technologies P-6000 spin coater was thenactivated to a spin speed of 7,000 rpm, and a spin time of 90 seconds.After the spinning was completed, the first layer was allowed to dry for15 minutes. The sensor 28 was then spun again at 7,000 rpm, and thesecond layer of the membrane material was applied. Two additional layerswere applied using the spin/flood technique used to apply the secondlayer, allowing 15 minutes between casting each layer. After all fourlayers have been cast, the membrane 94 was cured overnight at roomtemperature in a dust-free environment. The total thickness of themultiple layers of membrane 94, after curing, is approximately 20.0microns.

EXAMPLE II

Active layer 96 was prepared for use in working electrode 90 (as notedin Example I). The active layer 96, for a glucose sensor, primarilyincludes a catalytically active quantity of glucose oxidase, availablefrom Biozyme Laboratories, immobilized onto platinized carbon powderparticles, available from E-TEK, and the particles are distributedsubstantially uniformly throughout the layer.

About 3.15 grams of platinized carbon powder particles, Vulcan® XC-72carbon black, available from Cabot Corporation, Boston, Mass., preparedby the deposition of colloidal platinum (particle size between about 1.5to 2.5 nm) onto the surface of the carbon powder (nominal particle sizeabout 30 nm) by oxidative decomposition of complex platinum sulfite acid(II) using H₂ O₂, were treated in a phosphate buffer to neutralize anyresidual sulfuric acid present. The phosphate buffer also includes amicrobicide, sold under the trademark Kathon® CG microbicide of Rohm &Haas Corp., Philadelphia, Pa. The buffer was prepared by adding 11.499grams sodium phosphate, dibasic (Na₂ HPO₄), 2.898 grams sodiumphosphate, monobasic monohydrate (NaH₂ PO₄ "H₂ O), and 1.0 gram of theKathon® CG microbicide to 1.0 liter of distilled water. The bufferformulation was tested using a pH meter and electrode, to have a pH of7.5. Approximately 100 ml of the phosphate buffer was added to the 3.15grams of platinized activated carbon, and was mixed for 7 days. Thebuffer was replaced after the first 3 days of mixing by allowing theplatinized activated carbon to settle, decanting off 60 ml of the usedbuffer, and replacing it with 100 ml of fresh buffer. The mixture wasthen vacuum filtered after the 7 days of mixing, and the neutralizedcarbon was washed while under vacuum filtration using 100 ml of buffer.The vacuum was maintained for about 15 to 20 seconds after the bulk ofthe buffer had been pulled through the carbon to slightly dry the carbonand improve handling of the material.

The platinized activated carbon (PAC) was then mixed with 625 mg ofPentex® bovine serum albumin (BSA). The 625 mg of BSA was first added toa flask containing the PAC and an additional 40 ml of buffer. The BSAand PAC were gently mixed with a laboratory rotator and allowed to sitfor 1/2 hour to permit the BSA to dissolve. The mixture was again gentlymixed overnight at a speed setting of 3.5 for approximately 18 hours atroom temperature. The BSA-PAC mixture was then vacuum filtered andwashed under the vacuum filtration with 100 ml of buffer. Again, thevacuum was applied for about 20 seconds after the bulk of the buffer waspulled through the BSA-PAC to dry the BSA-PAC to between about 60 to 70percent moisture. The BSA-PAC was then refrigerated for future use inthe active and inactive layer inks for screen printing.

The active layer ink was formulated by adding 5.0 grams of a binderresin, available as product number 8101 RS from Metech, to 2.0 grams ofthe BSA-PAC (as prepared above). To this mixture, 0.25 gram of Triton®X-100 surfactant was added as a printing flow aid and wetting agent forthe layer. The mixture was then milled using a standard paint industrythree roll mill. 1.0 ml of AlbessoT thinner, available from Metech as8101 RS thinner, was added to the mixture, after the first milling wascompleted to adjust the viscosity of the paste for printing purposes.The mixture was then milled for a second period. Lastly, 0.4 gram ofglucose oxidase, available from Biozyme Laboratories, was added andmilled into the mixture. The active paste was then screen-printed ontoconductive strip 66 electrode portion to form working electrode 90.

EXAMPLE III

An inactive layer ink, used to form the interference correctingelectrode 92, was formulated using the procedure set forth in ExampleII. The inactive layer, however, does not include any catalyticallyactive quantity of an enzyme such as glucose oxidase. The inactive layerink was prepared by milling 5.0 grams of binder resin with 2.0 grams ofBSA-PAC (as prepared in Example II), 0.25 gram of Triton® X-100surfactant and 1.0 ml of AlbessoT (8101 RS) thinner. To this mixture anadditional 0.4 gram of Pentex® BSA was added and milled. Inactive layerpaste was then screen-printed onto conductive strip 70 electrode portionto form interference correcting electrode 92.

EXAMPLE IV

Referring again to FIGS. 1 through 8, one suitable construction of asensor package 10 including the components and design substantially inaccordance with an aspect of the present invention is provided by thefollowing combination of elements.

Sensor package 10 is molded of V825 acrylic plastic, available from Rohm& Haas Corp., and includes an open back J-body, having a width of about0.5 inch, a main body 14 length of about 1.535 inches, and a thicknessof about 0.37 inch. A handle 12, or gate portion, extends from the mainbody for aiding the insertion or removal of the sensor package 10 intoor from an instrument. The package 10 includes a housing 20 having asubstantially oval-shaped recess 22 formed therein. The recess has alength of about 1.0 inch and a width of about 0.065 inch. The recessincludes an outer perimeter 24 and a passageway 18 made up of an inlet56 and outlet 58. The passageway enters and exits recess 22 lengthwise.Passageway 18 has a substantially circular cross-section and a diameterof approximately 0.03 inch. As shown in FIGS. 5-8, a velocitycompensator or bump 60 is provided in the recess 22. Velocitycompensator 60 traverses the width of recess 22, and is approximately0.065 inch in length and about 0.04 inch in width. The velocitycompensator 60 is a bump-like protrusion in passageway 18 which has aradius of about 0.02 inch. The velocity compensator reduces the internalvolume of the sample chamber and creates a cross-sectional area close tothe inlet 56 and outlet 58 diameters. A gasket 26 is then provided tocontact, and form a seal between, the housing recess perimeter 24 and asensor 28 (as prepared in Example I). Gasket 26 is made from SarlinkT2450 elastomer having a hardness of about 50 on the Shore A scale.Gasket 26 is square-shaped, having sides of about 0.17 inch. Gasket 26further includes a substantially oval-shaped opening having a length ofabout 1.0 inch and a width of about 0.064 inch.

Gasket 26 is approximately 0.014 inch thick at its central cavityportion and approximately 0.05 inch thick at two outer sides along thelength of the gasket opening. These thicker surfaces allow the gasket tofit around the housing recess perimeter, while also allowing the recess22 to be open to the sensor electrode area to form a sensor samplechamber.

As noted in Example I a solid state, planar electrochemical sensor isformed on a ceramic substrate of about 0.025 inch thickness, and 0.45inch length and 0.18 inch width. The sensor 28 is placed upon a base padmade of a silicone rubber material having a hardness of between about 40to 60 on the Shore A scale. The pad has a length of about 0.5 inch and awidth of about 0.227 inch. The base pad 30 has a total thickness ofabout 0.058 inch, including a series of transverse protrusions on therear side thereof which extend about 0.015 inch from the base pad rearsurface and are spaced about 0.1 inch apart. The base pad 30 alsoincludes a central rectangular-shaped cavity on the opposite sidethereof for receipt of sensor 28. The cavity is about 0.45 inch long andabout 0.185 inch wide.

Lastly, a contact lead frame 32 is provided to connect sensor 28 to aninstrument which can measure and convert the current to determine theglucose (or lactate) concentration in the sample. Lead frame 32, alsoshown in FIGS. 3 and 4, includes four leads 34, each approximately 0.041inch wide at a base end and about 0.026 inch wide at the lead contacts50. The leads are approximately 1 inch in length and approximately 0.01inch thick. The leads 34 are made from a BeCu alloy material, which isnickel plated to a thickness of between about 40 to 80 microinches, andgold plated with a microelectronic grade gold plate material to athickness of between about 20 to 50 microinches thickness.

The lead frame 32, also molded of V825 acrylic plastic, includes theleads secured to a base 36 at a first end portion 38, and a sensorrecess 40 at a second end portion 42. The sensor recess 40 is about0.042 inch deep, approximately 0.5 inch in length, and about 0.225 inchin width, for receipt of the base pad 30 and sensor 28. Lead frame 32includes a second rectangularly-shaped recess 46 that is about 0.06 inchdeep, about 0.296 inch in length, and about 0.085 inch in width. Thesecond recess 46 is for receipt of a stabilizer bar 44, which aligns theleads 34 with the sensor contact pads (described above). The stabilizerbar 44 is a rectangular-solid shaped piece, also molded of the V825acrylic plastic material from Rohm & Haas Corp. The stabilizer bar isapproximately 0.29 inch in length and 0.075 inch in width and height.

After the base pad 30 and sensor 28 are placed into the sensor recess40, leads 34 are bent around frame 32 until leads 34 come into contactwith the sensor, and the stabilizer bar 44 is secured in recess 46. Thelead frame is approximately 1.147 inches in length and about 0.395 inchin width. After the components including the gasket 26, sensor 28, basepad 30 and contact lead frame 32 are assembled, the housing and leadframe are secured together by an ultrasonic weld around the outerperiphery of the contact lead frame. Four instrument electrical contactsurfaces 52 are exposed after the sensor package 10 is assembled. Thecontact surfaces are spaced between three dividers which extend pastlead frame first end 38 about 0.064 inch, and are about 1.0 inch longand 0.033 inch wide. Instrument contact surfaces 52 have about 1.0 inchexposed for electrical contact with an instrument.

EXAMPLE V

A planar glucose sensor, constructed substantially in accordance withEXAMPLES I-IV, was evaluated with whole blood, and the relationshipbetween glucose concentration in mg/dl and sensor current in nanoamperes(nA) was plotted as shown in FIG. 12. One of the significant features ofthe sensor, as graphically illustrated in FIG. 12, is the linearrelationship of glucose concentration to sensor current. It is believedthat the sensor membrane 94, being both glucose and oxygen-permeable,allows a stoichiometric excess of oxygen to glucose to permeate themembrane from whole blood resulting in the linear relationship from thelow end to the high end of the graph.

A similar sensor was evaluated to determine the response to lactate inwhole blood. The sensor used was substantially equivalent to thatconstructed in EXAMPLES I-IV, with the exception of the use of lactateoxidase instead of glucose oxidase. The relationship between the lactateconcentration in mmoles/L and sensor current in nanoamperes (nA) wasplotted in FIG. 13. One of the significant features of the sensor, asgraphically illustrated in FIG. 13, is the linear relationship oflactate concentration to sensor current. Once again, it is believed thatthe membrane, being both lactate and oxygen-permeable, allows astoichiometric excess of oxygen to lactate to permeate the membrane fromwhole blood resulting in the linear relationship from the low end atabout 100 mmoles/L to the high end of the graph at about 20.0 mmoles/Llactate.

EXAMPLE VI

To determine the effect of the interference correcting electrode 92, aglucose sensor response to glucose concentration, with and without thecorrecting electrode applied, was recorded as graphically illustrated inFIG. 14. Electrode 92 is provided to adjust for any interfering species,such as the neutral species acetaminophen, which can diffuse through thesensor's semi-permeable membrane 94.

In this example, 1.0 mmole/L of an interfering substance (acetaminophen)was added to a series of blood samples, covering a range of glucoseconcentrations up to 500.0 mg/dl. As noted, the data are shown in FIG.14 with and without the correcting electrode applied. Without thecorrecting electrode, there is approximately a 65.0 mg/dl positiveoffset from the case where the correcting electrode is applied. In otherwords, if the interference correcting electrode is not used, a meanerror of +65.0 mg/dl is obtained over the range of glucoseconcentrations. This is enough to cause a normal blood glucose level ofabout 82.0 mg/dl to read outside of the normal range, to about 47.0mg/dl if left uncorrected.

The glucose sensor response with the correcting electrode applied showsexcellent correlation with the ideal correction.

EXAMPLE VII

To determine the lifetime of the present electrochemical sensors, aglucose sensor, constructed substantially in accordance with EXAMPLESI-IV, was used over an extended sampling period, and for a large numberof samples. The sensor was tested over a period of sixty-nine (69) days,wherein a total of two thousand two hundred fifty (2,250) samples wereevaluated. The current in nA for an aqueous solution having a glucoseconcentration of 180 mg/dl was measured at various test points over thesixty-nine (69) day period. As shown in FIG. 15, the present glucosesensor provides a response over 10 nA for a period of at leastsixty-nine (69) days, and/or at least two thousand two hundred fifty(2,250) samples.

EXAMPLE VIII

To determine the effect of sensor post-treatment with surfactant on theinitial performance after a storage period, sensors were tested and therelationship between glucose concentration from about 83.0 mg/dl toabout 470.0 mg/dl and sensor current in nanoamperes (nA) was recorded inFIG. 18. A first sensor was post-treated with Triton® X-100 (as notedabove) while a second sensor was not post-treated. The sensors werestored one week at room temperature prior to the present evaluation. Theuntreated sensor exhibits a low and non-linear response to the glucoseconcentration (as shown more clearly in the exploded portion of thegraphical illustration shown in FIG. 18, the response of the untreatedsensor exhibits sensor drift past about 200.0 mg/dl glucose). This isthe result of slow wetup caused by the membrane drying out duringstorage. On the other hand, the treated sensor exhibits a linear, fullywetup response after only one hour of wetup.

A variety of surfactants were evaluated to determine the effect ofsensor post-treatment with a surfactant on the initial performance ofthe sensor. An aqueous sample having a glucose concentration of about180.0 mg/dl was tested with five glucose sensors. The first sensor hadno post-treatment, and the remaining sensors were separatelypost-treated with Triton® X-100 surfactant, Tergitol® 15 surfactant fromUnion Carbide Chemicals and Plastics Co., Inc., Danbury, Conn., Tween®20 ethoxylated sorbitan esters surfactant and polyethylene glycol havinga molecular weight of about 300. The sensors were tested and therelationship between the post-treatment and the sensor current innanoamperes (nA) was plotted in FIG. 17. In the absence of anypost-treatment, glucose sensors become difficult to wetup, as evidencedfrom the low response observed with untreated sensors. This effect isthe result of the membrane drying out during storage. Treatment of thesensor with an antidrying agent, such as the surfactants utilizedherein, more than doubled the sensor current output.

EXAMPLE IX

To determine the effect of membrane thickness on the linearity of asensor's response, a thin membrane 2-layer (about 10.0 microns), a thickmembrane 2-layer (about 22.0 microns), and a 4-layer membrane (about22.0 microns) were separately evaluated. The sensors were tested and therelationship between the glucose concentration in an aqueous solution inmg/dl and sensor current in nanoamperes (nA) was plotted in FIG. 19. Themulti-layer membranes were all prepared from anionically stabilized,water-based hydroxyl endblocked polydimethylsiloxane elastomercontaining about 14.0 percent by weight colloidal silica, commerciallyavailable as FC-61 coating from Dow Corning, Midland, Mich. As can beobserved from FIG. 19, multiple layers improve sensor performance asevidenced by a linear response. The thick 2-layer membranes of the samethickness (about 22.0 microns) as the 4-layer membranes exhibit higheroutput and a non-linear response to glucose. The 4-layer membraneprovides improved performance due to the elimination of membranedefects. The 2-layer membrane has membrane defects which allow an excessof glucose, with respect to oxygen, to pass through the membrane therebyaccounting for the non-linearity of the response, as well as the higheroutput.

A similar evaluation was performed comparing a 2-layer (about 11.0microns) and a 4-layer (about 18.0 microns) spin-cast membrane of FC-61coating material. The sensors were tested and the relationship betweenthe glucose concentration, ranging from about 69.0 mg/dl to about 487.0mg/dl, and sensor current in nanoamperes (nA) was plotted in FIG. 20.Again, the 2-layer spin-cast membrane comprised defects which allowed anexcess of glucose with respect to oxygen to permeate the membrane, whichresulted in higher output and a non-linear response.

A 2-layer (about 10.0 microns) spin-cast membrane was also compared to astenciled membrane (about 65.0 microns) to determine an effectivemembrane thickness. The membranes were comprised of the commerciallyavailable FC-61 coating material (as noted above). The sensors weretested and the relationship between the glucose concentration, up toabout 500.0 mg/dl, and the sensor current in nanoamperes (nA) wasplotted in FIG. 21. The thick, stenciled membranes exhibit slow wetup asevidenced by the non-linear glucose concentration response and the lowoutput. It was observed that membranes with a thickness of 65.0 micronsare too thick to provide for a useful glucose response. Note thepositive deviation from a linear response of the stenciled film in theinset graph. Moreover, the thick stenciled membranes had a slow responsetime of greater than about 60 seconds. The 2-layer, spin-cast membraneexhibits high output and non-linear response (as described above).

EXAMPLE X

To determine the effect of incorporating a surfactant in the platinizedactivated carbon (PAC) material on performance versus storage, a glucosesensor response was evaluated with no surfactant in the PAC after oneday and after 21 days in storage at room temperature. The sensors weretested and the relationship between glucose concentration, up to about500.0 mg/dl, and sensor current in nanoamperes (nA) was plotted in FIG.22. As shown in FIG. 22, the sensor output degrades over time if asurfactant, such as Triton® X-100, is not added to the PAC material.FIG. 16 shows the same effect as FIG. 22 but with the optimizedmulti-layer membrane.

FIG. 23 is a graphical illustration of the glucose concentration, up toabout 500.0 mg/dl, and sensor current in nanoamperes (nA) wherein theglucose sensors include Triton® X-100 surfactant in the PAC material.The addition of the surfactant to the PAC, active and inactive layers,aids in sensor wetup of aged sensors. The addition of the surfactant inthe PAC material provides for equivalent performance in new and agedsensors.

EXAMPLE XI

To determine the effect of adding a surfactant to membrane 94 on thesensors performance, glucose sensors were tested and the relationshipbetween glucose concentration, up to about 500.0 mg/dl, and sensorcurrent in nanoamperes (nA) was plotted in FIG. 24. The sensor membrane94, comprised essentially of the commercially available FC-61 coatingmaterial (as described above) was applied to separate sensors; onesensor also included a surfactant material, Makon® 10 surfactantavailable from Stepan Co., Northfield, Ill. Both membranes were 2-layer,spin-cast membranes about 11.0 microns thick. The addition of asurfactant in the membrane provides improved wetup and higher responseto glucose concentration. This effect can be minimized, although noteliminated if membranes are post-treated with an antidrying agent, asshown in FIGS. 17 and 18.

Although particular embodiments of the invention have been described indetail for purposes of illustration, various modifications may be madewithout departing from the spirit and scope of the present invention.Design considerations may alter the configuration of the sensor and/orthe sensor package to optimize the efficiency of certain applicationsand minimize the cost associated with the production and use thereof.Accordingly, this invention is not to be limited except by the appendedclaims.

                                      TABLE I                                     __________________________________________________________________________    SCREEN PRINTED LAYERS                                                         LAYER                                                                             INK            OVEN/FURNACE                                                                           TEMP./RECIPE                                      __________________________________________________________________________    1   GOLD           FURNACE  STD-850                                           2   PLATINUM       FURNACE  PTDI-750                                          3   SILVER         FURNACE  PTDI-750                                          4   DIELECTRIC     FURNACE  PTDI-750                                          5   Ag/AgCl        OVEN     75° C. 30 MINUTES                          6   CELLULOSE ACETATE                                                                            OVEN     55° C. 10 MINUTES                                                      RAMP TO 100° C. 10                                                     MINUTES 100° C. 10                                                     MINUTES                                           7   BSA-PAC (INACTIVE)                                                                           OVEN     55° C. 20 MINUTES                          8   GLUCOSE OXIDASE (ACTIVE)                                                                     OVEN     55° C. 20 MINUTES                          __________________________________________________________________________

What is claimed is:
 1. A sensor comprising a semi-permeable membranewhich permits passage of water, oxygen and at least one selectiveanalyte and which has the ability to absorb water, for contacting a testsample, said membrane having a high boiling point, water soluble,hydrophilic polymer liquid anti-drying agent which is added after saidmembrane has been formed so that the sensor storage life is prolongedand wet-up is facilitated.
 2. The sensor of claim 1, wherein saidanti-drying agent comprises a material selected from the groupconsisting of surfactants having a molecular weight of at least about300 and polyethylene glycols having molecular weights of between about200 and
 600. 3. A sensor in accordance with claim 2 wherein saidanti-drying agent is a polyethylene glycol.
 4. A sensor in accordancewith claim 3 wherein said membrane is an anionically stabilized, waterbased hydroxyl end blocked polydimethyl siloxane elastomer containing atleast 10% of weight of silica.
 5. A sensor in accordance with claim 1wherein said membrane is a polydimethyl siloxane.
 6. A sensor inaccordance with claim 1 wherein said membrane is a polydimethyl siloxaneelastomer containing at least 10% by weight of silica.
 7. A method forprolonging the storage life or wet-up of an electrochemical sensorincluding a semi-permeable membrane, said method comprising treating thesemi-permeable membrane after formation with a high boiling point, watersoluble, hydrophilic polymer liquid anti-drying agent selected from thegroup consisting of surfactants having a molecular weight of at leastabout 300 and polyethylene glycols.